The glycopeptide antibiotic vancomycin has been the last defence against antibiotic-resistant bacteria for the past 40 years. The recent emergence of vancomycin-resistant bacteria is thus a very serious health problem. Efforts to synthesize more active analogs of vancomycin are limited by the drug's structural complexity. The long-term goal of the proposed research is the engineered biosynthesis of new glycopeptides. The genes needed for biosynthesis of the glycopeptide chloroeremomycin (CE) have recently been sequenced, revealing that glycopeptides derive from peptide precursors synthesized by non-ribosomal peptide synthases (NRPSs). For NRPSs, the sequence of protein domains determines the sequence of the peptide product, suggesting that domain-swapping could provide new glycopeptides. The proposed research will use recently characterized phosphopantetheinyl transferase enzymes to prepare active fragments (both native and hybrid) of the CE synthase following expression in E. coli. These constructs will be used to examine the breadth of substrate specificity of peptide bond-forming and product release domains within the synthase, a key step toward the engineered biosynthesis of new potent glycopeptides.